Drugs that generate DNA crosslinks are among the most effective cancer chemotherapeutic agents. These drugs fall in several classes of bi-functional molecules that generate DNA mono-adducts, intrastrand and interstrand crosslinks and are the standard of care for many malignancies. Interstrand crosslinks (ICLs) block many DNA transactions and are thought to be the cytotoxic lesions responsible for most crosslinking drug efficacy. Their efficacy however, varies among cell types or patients. There are two major challenges associated with these compounds: 1) dose-limiting toxicity, primarily in the blood, and 2) acquired resistance. The goal of this proposal is to characterize the mechanisms of ICL repair to better understand and ultimately improve the mode of action of crosslinking agent-based chemotherapy. ICLs are repaired during and outside of S-phase. Replication-independent ICL repair (RIR) is robust and critical for survival in treated mammalian cells. The proposed studies specifically address the mechanism of ICL repair in the absence of other DNA lesions and therefore focus on the most clinically relevant repair reaction triggered by crosslinking drugs. Specifically, we propose to identify the nucleases (Aim 1) and DNA polymerases (Aim 2) involved in RIR. Finally, we propose to evaluate the impact of replication-dependent and -independent ICL repair on crosslinking drugs efficacy. We hypothesize that targeting these factors could increase the sensitivity of tumor cells to crosslinking agents and reduce the incidence of resistance. Our approach will combine biochemistry in cell-free extracts with innovative ICL repair assays in normal and tumor cells. We anticipate that a better understanding of the molecular mechanisms of ICL repair will shed light on the contribution of ICL lesions to crosslinking drug toxicity and on the impact of DNA repair on resistance to crosslinking therapy.